Human CD4⁺CD25^hiFoxP3⁺ Treg cells induce senescence in naive CD4⁺ T cells. (A) CD4⁺CD25^hi FoxP3⁺ Treg cell treatment increased SA-β-Gal⁺ T-cell populations in naive CD4⁺ T cells significantly. Naive CD4⁺ T cells cultured in medium only or cocultured with CD4⁺CD25⁻ effector T cells had little or no SA-β-Gal expression. Naive CD4⁺ T cells treated with ionizing radiation (5 Gy X-ray) served as positive controls exhibiting SA-β-Gal expression. CFSE-labeled naive CD4⁺ T cells were incubated alone or were cocultured with Treg cells or CD4⁺CD25⁻ T cells at a ratio of 5:1 in the presence of plate-bound anti-CD3 (2 μg/mL) for 5 days. The treated naive CD4⁺ T cells were purified by FACS and stained with SA-β-Gal staining reagents after an additional 3-day culture. The SA-β-Gal⁺ T cells were identified with dark blue granules, as indicated by the arrows. (B) Time course of the induction of the SA-β-Gal⁺ T-cell populations in Treg-treated naive CD4⁺ T cells. The percentages of SA-β-Gal⁺ cells in naive CD4⁺ T cells treated with CD4⁺CD25^hi FoxP3⁺ Treg cells were increased dramatically with progressive coculture time. Cell treatment and procedure were the same as in panel A. (C) Treatment with CD4⁺CD25^hi FoxP3⁺ Treg cells inhibited naive T-cell growth and resulted in decreased cell numbers with increasing coculture time. Cell treatment and procedure were the same as in panel A and cell numbers were counted by FACS gated on the CFSE⁺ population. (D) Decreased expression of CD27 and CD28 in naive CD4⁺ T cells treated by CD4⁺CD25^hi FoxP3⁺ Treg cells. Cell treatment and procedure were the same as in panel A. Naive CD4⁺ T cells treated with ionizing radiation (5 Gy X-ray) served as positive controls. CD27 and CD28 expression in treated naive CD4⁺ T cells were analyzed by FACS gating on CFSE⁺ populations. (E) The cytokines IL-2, IL-7, and IL-15 cannot prevent senescence identified by SA-β-Gal expression in naive CD4⁺ T-cell populations induced by CD4⁺CD25^hi FoxP3⁺ Treg cells. Naive CD4⁺ T cells were cocultured with CD4⁺CD25^hiFoxP3⁺ Treg cells for 3 days in the presence of various concentrations of the indicated cytokines. The treated naive CD4⁺ T cells were purified and SA-β-Gal expression was determined.